Ratiometric Near-Infrared Fluorescence Liposome Nanoprobe for H2S Detection In Vivo

Accurate detection of H2S is crucial to understanding the occurrence and development of H2S-related diseases. However, the accurate and sensitive detection of H2S in vivo still faces great challenges due to the characteristics of H2S diffusion and short half-life. Herein, we report a H2S-activatable ratiometric near-infrared (NIR) fluorescence liposome nanoprobe HS-CG by the thin-film hydration method. HS-CG shows “always on” fluorescence signal at 816 nm and low fluorescence signal at 728 nm; the NIR fluorescence ratio between 728 and 816 nm (F728/F816) is low. Upon reaction with H2S, the fluorescence at 728 nm could be more rapidly turned on due to strong electrostatic interaction between enriched HS− and positively charged 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) doped in the liposome nanoprobe HS-CG, resulting in a large enhancement of F728/F816, which allows for sensitive visualization of the tumor H2S levels in vivo. This study demonstrates that this strategy of electrostatic adsorption between HS− and positively charged molecules provides a new way to enhance the reaction rate of the probe and H2S, thus serving as an effective platform for improving the sensitivity of imaging.


Introduction
Hydrogen sulfide (H 2 S), the third endogenous gaseous transmitter besides nitric oxide (NO) and carbon monoxide (CO), is produced from a cysteine substrate or its derivatives and catalyzed by several enzymes such as cystathionine g-lyase (CSE), cystathionine b-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase [1][2][3]. H 2 S plays an important role in biological functions and mediating many biological processes including cytoprotection [4,5], mediation of neurotransmission [6,7], vascular tone regulation [8,9], anti-inflammation effect [10,11], etc. However, abnormal levels of H 2 S are correlated with the symptoms of numerous diseases such as Down syndrome, Alzheimer's disease, and cancers [12][13][14][15][16]. Therefore, the highly sensitive and specific accurate detection of H 2 S levels in biological systems is of great significance for understanding H 2 S relevant physiological and pathological processes.
Ratiometric fluorescence imaging is a method where intensities at two or more different wavelengths are measured to detect the dynamic behavior and function relationships of biomolecules in living organisms [17][18][19]. Ratiometric fluorescence imaging provides builtin self-calibration for factors unrelated to the analyte (e.g., variable probe concentration and Figure 1 shows the design of the NIR ratiometric fluorescence liposome nanoprobe (HS-CG). HS-CG was prepared by 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC)-, cholesterol-, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-mPEG2000)-assisted encapsulating of H 2 S activatable IR-HS and indocyanine green (ICG) through thin-film hydration method. IR-HS is used as an imaging reagent because we previously proved that it has excellent stability and can produce bright NIR fluorescence at 728 nm under 680 nm excitation when IR-HS reacts with H 2 S rapidly ( Figure 1a) [38]. ICG is used as a fluorescent dye because of its stable and bright NIR fluorescence at 816 nm [39,40], which can avoid interference from the fluorescence of dye 1 at 728 nm and use as a reliable internal reference for H 2 S detection. Initially, the liposome nanoprobe HS-CG displays the "always on" bright NIR fluorescence signal of ICG at 816 nm and the "off" fluorescence signal of IR-HS at 728 nm. The fluorescence intensity ratio between 728 and 816 nm (F 728 /F 816 ) is very low. Upon reaction with H 2 S, IR-HS within HS-CG is converted into fluorescence dye 1 with fluorescence emission at 728 nm, and ICG's fluorescence at 816 nm is unchanged, resulting in an increase in the fluorescence ratio F 728 /F 816 , which would be suitable for detecting H 2 S levels. The ratiometric NIR fluorescence liposome nanoprobe HS-CG possesses many unique advantages: (1) Due to the doping of DPPC, the positive charge on the surface of liposome nanoparticles HS-CG can effectively adsorb HS − with a negative charge, which improves the reaction rate between IR-HS within HS-CG and H 2 S; (2) the "always on" NIR fluorescence signal of ICG at 816 nm of the liposome nanoparticles HS-CG prepared by DPPC and DSPE-mPEG2000 is significantly higher than that of micellar nanoparticles HS-CG NP prepared by DSPE-mPEG2000, which improves the sensitivity of tumor detection; and (3) the NIR fluorescence intensity ratio F 728 /F 816 provided by HS-CG can avoid the interference of factors unrelated to the analyte such as the concentration of the HS-CG and environmental factors, and improve the reliability and sensitivity of H 2 S detection. With these advantages of liposome nanoparticles HS-CG, we used the "always on" and DSPE-mPEG2000 is significantly higher than that of micellar nanoparticles HS-CG NP prepared by DSPE-mPEG2000, which improves the sensitivity of tumor detection; and (3) the NIR fluorescence intensity ratio F728/F816 provided by HS-CG can avoid the interference of factors unrelated to the analyte such as the concentration of the HS-CG and environmental factors, and improve the reliability and sensitivity of H2S detection. With these advantages of liposome nanoparticles HS-CG, we used the "always on" fluorescence signal at 816 nm to noninvasively track the delivery and accumulation of HS-CG in the tumors after systemic administration while the activatable fluorescence signal at 728 nm was used to reliably detect tumor endogenous H2S in mice.

Synthesis and Characterization of Liposome Nanoprobe HS-CG
We first synthesized the liposome nanoprobe HS-CG by the thin-film hydration method. UV-Vis-NIR adsorption spectra demonstrated characteristic absorption peaks of ICG and IR-HS, with encapsulation efficiencies of nearly 100%. The mass ratio of DPPC, cholesterol, DSPE-mPEG2000, ICG, and IR-HS was 4:2:4:0.035:0.027. These results proved the successful preparation of the liposome nanoprobe HS-CG ( Figure 2a). Dynamic light scattering (DLS) analysis demonstrated that liposome nanoprobe HS-CG could well disperse in an aqueous solution. The hydrodynamic size of the liposome nanoprobe HS-CG was found to be ~100 nm (Figure 2b). Transmission electron microscopy (TEM) analysis showed that the nanoprobe HS-CG appeared as a vesicle shape (Figure 2b, inset). UV-Vis-NIR adsorption spectra demonstrated that the absorption of HS-CG between 462 and 636 nm declined, and the absorption between 636 nm and 752 nm obviously increased after the reaction with NaHS ( Figure S1). Moreover, the fluorescence spectra of HS-CG initially had a NIR fluorescence emission at 816 nm ( Figure 2c). Upon reaction with NaHS, the fluorescence of the HS-CG at 728 nm was significantly enhanced by ~53-fold, while the fluorescence of the HS-CG at 816 nm remained unchanged. To verify the role of DPPC in the liposome nanoprobe HS-CG, we prepared the micellar nanoparticles (HS-CG NPs) with DSPE-PEG-assisted encapsulation of ICG and IR-HS. DLS showed that the particle

Synthesis and Characterization of Liposome Nanoprobe HS-CG
We first synthesized the liposome nanoprobe HS-CG by the thin-film hydration method. UV-Vis-NIR adsorption spectra demonstrated characteristic absorption peaks of ICG and IR-HS, with encapsulation efficiencies of nearly 100%. The mass ratio of DPPC, cholesterol, DSPE-mPEG2000, ICG, and IR-HS was 4:2:4:0.035:0.027. These results proved the successful preparation of the liposome nanoprobe HS-CG ( Figure 2a). Dynamic light scattering (DLS) analysis demonstrated that liposome nanoprobe HS-CG could well disperse in an aqueous solution. The hydrodynamic size of the liposome nanoprobe HS-CG was found to be~100 nm ( Figure 2b). Transmission electron microscopy (TEM) analysis showed that the nanoprobe HS-CG appeared as a vesicle shape (Figure 2b, inset). UV-Vis-NIR adsorption spectra demonstrated that the absorption of HS-CG between 462 and 636 nm declined, and the absorption between 636 nm and 752 nm obviously increased after the reaction with NaHS ( Figure S1). Moreover, the fluorescence spectra of HS-CG initially had a NIR fluorescence emission at 816 nm ( Figure 2c). Upon reaction with NaHS, the fluorescence of the HS-CG at 728 nm was significantly enhanced by~53-fold, while the fluorescence of the HS-CG at 816 nm remained unchanged. To verify the role of DPPC in the liposome nanoprobe HS-CG, we prepared the micellar nanoparticles (HS-CG NPs) with DSPE-PEG-assisted encapsulation of ICG and IR-HS. DLS showed that the particle size of the HS-CG NP was~52 nm, and the TEM showed that the HS-CG NPs had a spherical micelle morphology (Figure 2d). Fluorescence spectra showed that the HS-CG NPs had a fluorescence peak at 816 nm. When HS-CG NP reacted with NaHS, the fluorescence at 728 nm increased by 36-fold (Figure 2e), which indicated that the fluorescence activation ratio at 728 nm of the HS-CG NPs was significantly lower than that of the liposome nanoparticles HS-CG. Moreover, it is noteworthy that the fluorescence intensity of the micellar nanoparticles HS-CG NPs at 816 nm was 1.8-fold lower than that of the liposome nanoparticles HS-CG. Additionally, we showed that when the liposome nanoparticles HS-CG and micelle nanoparticles HS-CG NP with the same concentration were incubated with NaHS (1 mM), the fluorescence intensity of the liposome nanoparticles HS-CG reached a plateau at 200 s, 141 s earlier than that of the micelle nanoparticles HS-CG NP (Figure 2f), which was due to the positively charged surface of the liposome nanoparticles HS-CG with doping DPPC possibly accelerating the reaction between HS-CG and H 2 S. In addition, we found that the H 2 S-activated increment of the fluorescence ratio (F 728 /F 816 ) could be similarly acquired in different pH PBS buffers (6.0-8.0) ( Figure S2). Taken together, liposome nanoparticles HS-CG could rapidly react with H 2 S, and the fluorescence signal at 728 nm was significantly enhanced, thus effectively improving the sensitivity of H 2 S detection.
ical micelle morphology (Figure 2d). Fluorescence spectra showed that the HS-CG NPs had a fluorescence peak at 816 nm. When HS-CG NP reacted with NaHS, the fluorescence at 728 nm increased by 36-fold (Figure 2e), which indicated that the fluorescence activation ratio at 728 nm of the HS-CG NPs was significantly lower than that of the liposome nanoparticles HS-CG. Moreover, it is noteworthy that the fluorescence intensity of the micellar nanoparticles HS-CG NPs at 816 nm was 1.8-fold lower than that of the liposome nanoparticles HS-CG. Additionally, we showed that when the liposome nanoparticles HS-CG and micelle nanoparticles HS-CG NP with the same concentration were incubated with NaHS (1 mM), the fluorescence intensity of the liposome nanoparticles HS-CG reached a plateau at 200 s, 141 s earlier than that of the micelle nanoparticles HS-CG NP (Figure 2f), which was due to the positively charged surface of the liposome nanoparticles HS-CG with doping DPPC possibly accelerating the reaction between HS-CG and H2S. In addition, we found that the H2S-activated increment of the fluorescence ratio (F728/F816) could be similarly acquired in different pH PBS buffers (6.0-8.0) ( Figure S2). Taken together, liposome nanoparticles HS-CG could rapidly react with H2S, and the fluorescence signal at 728 nm was significantly enhanced, thus effectively improving the sensitivity of H2S detection.

Response of Liposome Nanoprobe HS-CG toward H 2 S In Vitro
To detect the sensitivity of liposome nanoprobe HS-CG toward H 2 S, the fluorescence spectra of HS-CG after incubation with different concentrations of NaHS (0, 1, 2, 5, 10, 15, 20, 50, 80, 100, 150, 200, 300, 400, 500 µM) for 6 min were acquired. Figure 3a-d showed that the fluorescence of HS-CG at 728 nm increased with the concentration of NaHS, while the fluorescence at 816 nm remained unchanged. The fluorescence ratio (F 728 /F 816 ) versus NaHS concentration from 0 to 20 µM showed a good linear correlation; the detection limit (LOD) of HS-CG toward H 2 S was determined to be~0.13 µM (Figure 3e). To evaluate the selectivity of HS-CG toward H 2 S, HS-CG was incubated with NaHS and other reducing agents (e.g., GSH, VC, L -Cys, Hcy). Figure 3f-i demonstrates that NaHS and other reductants (e.g., GSH, VC, L -Cys, Hcy) containing NaHS could activate the fluorescence of HS-CG at 728 nm, which resulted in a significant enhancement in the fluorescence ratio F 728 /F 816 (Figure 3h). However, the fluorescence spectra and fluorescence ratio F 728 /F 816 were unchanged for HS-CG following treatment with the other reductants, showing that the liposome nanoprobe HS-CG was highly specific for H 2 S. Additionally, we showed that liposome HS-CGs were quite stable in PBS buffer (pH 7.4) for 5 days, with little change in the fluorescence of IR-HS and ICG and fluorescence ratio F 728 /F 816 upon incubation (PBS, pH 7.4) for 120 h ( Figure S3). These results demonstrate that liposome HS-CG could serve as an efficient ratiometric imaging probe in vivo.
F728/F816 (Figure 3h). However, the fluorescence spectra and fluorescence ratio F728/F816 were unchanged for HS-CG following treatment with the other reductants, showing that the liposome nanoprobe HS-CG was highly specific for H2S. Additionally, we showed that liposome HS-CGs were quite stable in PBS buffer (pH 7.4) for 5 days, with little change in the fluorescence of IR-HS and ICG and fluorescence ratio F728/F816 upon incubation (PBS, pH 7.4) for 120 h ( Figure S3). These results demonstrate that liposome HS-CG could serve as an efficient ratiometric imaging probe in vivo.

Imaging of H2S in Tumor Cells
Prior to cell ratiometric imaging, the cell viability of the liposome nanoprobe HS-CG was first evaluated via the MTT analysis. Figure S4 shows that HS-CG had excellent biocompatibility for cells during the incubation of HCT116 colorectal cancer cells or human embryonic kidney cells (HEK293 cells) with HS-CG for 24 h. To optimize the incubation time and concentration, we then utilized the "always on" fluorescence of ICG within HS-CG to examine the uptake of HS-CG in the HCT116 cells. We showed that the fluorescence of ICG in cells increased in a time-and concentration-dependent manner (Figures S5 and  S6). The ICG fluorescence reached the maximum in cells following incubation with liposome nanoprobe HS-CG at a concentration of 4 μM for 3 h. Moreover, we found that the

Imaging of H 2 S in Tumor Cells
Prior to cell ratiometric imaging, the cell viability of the liposome nanoprobe HS-CG was first evaluated via the MTT analysis. Figure S4 shows that HS-CG had excellent biocompatibility for cells during the incubation of HCT116 colorectal cancer cells or human embryonic kidney cells (HEK293 cells) with HS-CG for 24 h. To optimize the incubation time and concentration, we then utilized the "always on" fluorescence of ICG within HS-CG to examine the uptake of HS-CG in the HCT116 cells. We showed that the fluorescence of ICG in cells increased in a time-and concentration-dependent manner (Figures S5 and S6). The ICG fluorescence reached the maximum in cells following incubation with liposome nanoprobe HS-CG at a concentration of 4 µM for 3 h. Moreover, we found that the fluorescence of HS-CG could well coincide with the fluorescence of lysosomes in cells, indicating that HS-CG could effectively locate the lysosome of the cells ( Figure S7).
With the optimized incubation conditions, the ratiometric fluorescence imaging of H 2 S in living cells was conducted following incubation with HS-CG (4 µM, 3 h). Figure 4a-c shows that HCT116 cells incubated with HS-CG possessed bright fluorescence under both fluorescence dye 1 and the ICG channels, and the intracellular fluorescence ratio F IR-HS /F ICG was measured was~0.21 ± 0.07. When ZnCl 2 (an H 2 S scavenger) was added to the cells, the intracellular fluorescence of IR-HS was reduced, while the fluorescence of ICG remained unchanged. Therefore, the F IR-HS /F ICG was reduced to~0.06 ± 0.03. When NaHS was added to cells to upregulate the concentration of exogenous H 2 S in the cells, the F IR-HS /F ICG was obviously enhanced to~1.0 ± 0.1 because the fluorescence of IR-HS was activated, which was decreased by ZnCl 2 . HS-CG was then applied to detect endogenous H 2 S generation in HCT116 cells. Upon the incubation with L -Cys (endogenous substrate of CSE and CBS for H 2 S production), the fluorescence of IR-HS in the cells became brighter and the F IR-HS /F ICG was obviously~4.6-fold higher than that of the untreated cells (Control group), which could be effectively blocked by the CSE inhibitor propargylglycine (PAG) and CBS inhibitor aminooxyacetic acid (AOAA) (Figure 4d). These image results show that HS-CG could monitor the H 2 S levels in cells via ratiometric fluorescence imaging.
With the optimized incubation conditions, the ratiometric fluorescence imaging of H2S in living cells was conducted following incubation with HS-CG (4 μM, 3 h). Figure  4a-c shows that HCT116 cells incubated with HS-CG possessed bright fluorescence under both fluorescence dye 1 and the ICG channels, and the intracellular fluorescence ratio FIR-HS/FICG was measured was ~0.21 ± 0.07. When ZnCl2 (an H2S scavenger) was added to the cells, the intracellular fluorescence of IR-HS was reduced, while the fluorescence of ICG remained unchanged. Therefore, the FIR-HS/FICG was reduced to ~0.06 ± 0.03. When NaHS was added to cells to upregulate the concentration of exogenous H2S in the cells, the FIR-HS/FICG was obviously enhanced to ~1.0 ± 0.1 because the fluorescence of IR-HS was activated, which was decreased by ZnCl2. HS-CG was then applied to detect endogenous H2S generation in HCT116 cells. Upon the incubation with L-Cys (endogenous substrate of CSE and CBS for H2S production), the fluorescence of IR-HS in the cells became brighter and the FIR-HS/FICG was obviously ~4.6-fold higher than that of the untreated cells (Control group), which could be effectively blocked by the CSE inhibitor propargylglycine (PAG) and CBS inhibitor aminooxyacetic acid (AOAA) (Figure 4d). These image results show that HS-CG could monitor the H2S levels in cells via ratiometric fluorescence imaging.

Imaging of H 2 S in Tumor Cells
Encouraged by the results of cell imaging, we then utilized the HS-CG to conduct ratiometric fluorescence imaging for reporting the exogenous H 2 S levels in vivo. Mice with subcutaneous (s.c.) injection of HS-CG showed dark fluorescence at the IR-HS channel, and bright fluorescence at the ICG channel (Figure 5a,b). The fluorescence ratio F IR-HS /F ICG was low (Figure 5c). However, the fluorescence signal at the IR-HS channel was significantly enhanced in living mice with s.c. injection of HS-CG plus NaHS, while ICG fluorescence remained unchanged, resulting in the F IR-HS /F ICG ratio obviously being enhanced by~5.1-fold (Figure 5a-c). When ZnCl 2 was s.c. injected into mice treated with NaHS, the fluorescence of IR-HS decreased, with the F IR-HS /F ICG reduced by~1.9-fold (Figure 5a-c). These results indicate that HS-CG could serve as a ratiometric fluorescence imaging nanoprobe for monitoring exogenous H 2 S production in vivo.
with subcutaneous (s.c.) injection of HS-CG showed dark fluorescence at the IR-HS channel, and bright fluorescence at the ICG channel (Figure 5a,b). The fluorescence ratio FIR-HS/FICG was low (Figure 5c). However, the fluorescence signal at the IR-HS channel was significantly enhanced in living mice with s.c. injection of HS-CG plus NaHS, while ICG fluorescence remained unchanged, resulting in the FIR-HS/FICG ratio obviously being enhanced by ~5.1-fold (Figure 5a-c). When ZnCl2 was s.c. injected into mice treated with NaHS, the fluorescence of IR-HS decreased, with the FIR-HS/FICG reduced by ~1.9-fold (Figure 5a-c). These results indicate that HS-CG could serve as a ratiometric fluorescence imaging nanoprobe for monitoring exogenous H2S production in vivo. When HS-CG was intravenous (i.v.) injected into s.c. HCT116 tumor-bearing mice, the "always-on" ICG fluorescence images showed that HS-CG could gradually accumulate in tumors ( Figure S8a). Fluorescence intensities gradually increased with time and reached the maximum at 12 h ( Figure S8b). Ex vivo fluorescence images of the main organs and tissues at 12 h showed that the fluorescence of the tumors was significantly higher than that of other organs (e.g., heart, liver, spleen, lung, kidneys, intestines, stomach), which revealed that the HS-CG mainly accumulated in tumor at 12 h ( Figure S8c,d). Moreover, the biodistribution analysis also indicated that HS-CG mainly accumulated in the tumor (%ID/g ≈ 17.7%) and liver (%ID/g ≈ 10.2%) at 12 h post i.v. injection into s.c. HCT116 tumor-bearing mice ( Figure S8e). Next, we utilized HS-CG for noninvasive ratiometric fluorescence imaging of endogenous H2S generation in the HCT116 tumors of When HS-CG was intravenous (i.v.) injected into s.c. HCT116 tumor-bearing mice, the "always-on" ICG fluorescence images showed that HS-CG could gradually accumulate in tumors ( Figure S8a). Fluorescence intensities gradually increased with time and reached the maximum at 12 h ( Figure S8b). Ex vivo fluorescence images of the main organs and tissues at 12 h showed that the fluorescence of the tumors was significantly higher than that of other organs (e.g., heart, liver, spleen, lung, kidneys, intestines, stomach), which revealed that the HS-CG mainly accumulated in tumor at 12 h ( Figure S8c,d). Moreover, the biodistribution analysis also indicated that HS-CG mainly accumulated in the tumor (%ID/g ≈ 17.7%) and liver (%ID/g ≈ 10.2%) at 12 h post i.v. injection into s.c. HCT116 tumor-bearing mice ( Figure S8e). Next, we utilized HS-CG for noninvasive ratiometric fluorescence imaging of endogenous H 2 S generation in the HCT116 tumors of mice. When the HS-CG was i.v. injected into mice, the fluorescence in the IR-HS channel was significantly enhanced, and bright fluorescence was displayed in the ICG channel (Figure 5d,e); the fluorescence ratio F IR-HS /F ICG was determined to be~0.52 ± 0.06 (Figure 5f). The fluorescence of the tumor IR-HS was remarkably increased (∼1.9-fold) with intratumoral (i.t.) injection of L -Cys, while the fluorescence of ICG showed no obvious change. The F IR-HS /F ICG of the tumors increased~1.9-fold after 12 h, which was obviously reduced when the CSE activity was inhibited by PAG and AOAA (Figure 5f). These results demonstrate that HS-CG could reliably be used in the ratiometric imaging of tumors with endogenous H 2 S levels in living mice.
Absorption and fluorescence spectra were performed on an Ocean Optics UV-Visible spectrometer (Ocean Optics, Dunedin, FL, USA) and HORIBA Jobin Yvon Fluoromax-4 fluorescence spectrometer (HORIBA Jobin Yvon, Paris, France), respectively. Fluorescence images were acquired on an Olympus IX73 fluorescent inverted microscope (Olympus LS, Tokyo, Japan). Dynamic light scattering (DLS) was conducted on a 90 Plus/BI-MAS equipment (Brookhaven, New York, NY, USA). Transmission electron microscopy (TEM) analysis was performed on a JEM-1011 transmission electron microscope (JEOL, Ltd., Tokyo, Japan). The MTT analysis was conducted on a microplate reader (Tecan, Grödig, Austria). The fluorescence images in mice were collected using an IVIS Lumina XR III system (PerkinElmer, Waltham, MA, USA).

Preparation of Liposome Nanoprobe HS-CG
The liposome nanoprobe HS-CG was prepared through a thin-film hydration method. Briefly, IR-HS (0.027 mg), ICG (0.035 mg), DPPC (4 mg), cholesterol (2 mg), and DSPE-PEG2000-OMe (4 mg) were dissolved in a mixture of CH 3 CH 2 OH (1 mL) and DMSO (5 µL) to form a homogeneous solution. Then, we removed the organic solvent to form a thin layer by rotary evaporator. Subsequently, we added D.I. water (9 mL) into a thin layer under sonication. The remaining aqueous solution was further sonicated for 5 min and then passed through 0.22 µm filters three times. Finally, the heterogeneous liposome nanoprobe HS-CG was formed. The HS-CG was washed three times under ultrafiltration. The final solution was kept in the dark at 4 • C.

Cell Culture
Colon cancer cells (HCT116 cells) were grown in McCoy s 5A medium. The medium was supplemented with 10% (v/v) fetal bovine serum (FBS) and 100 units/mL penicillin plus 100 µg/mL streptomycin. HCT116 cells were kept in a humidified incubator containing 5% CO 2 at 37 • C.

Ratiometric Imaging of H 2 S in Living Tumor Cells
HCT116 cells were inoculated in a glass-bottom plate. After 12 h, the cells were incubated with HS-CG (4/4 µM IR-HS/ICG) for 3 h and washed with PBS buffer (1×), and then incubated with or without NaHS (1 mM) for 1 h. To eliminate the endogenous H 2 S, cells were pretreated with ZnCl 2 (300 µM) for 10 min. To eliminate the exogenous H 2 S, cells were pretreated with ZnCl 2 (300 µM) for 10 min, and cells were treated with NaHS (1 mM) for 1 h. To increase the endogenous H 2 S level, HCT116 cells were treated with L -Cys (200 µM) for 1 h. To inhibit the cystathionine γ-lyase (CSE) and cystathionine b-synthase (CBS) activity, cells were pretreated with DL-propargylglycine (PAG, 50 mg/L) plus aminooxyacetic acid (AOAA, 20 µM) for 0.5 h, and then treated with L -Cys (200 µM) for 1 h. After each treatment, cells were incubated with HS-CG (4/4 µM IR-HS/ICG) for 3 h. The fluorescence images were acquired on an Olympus IX73 fluorescent inverted microscope.

Animals and Tumor Models
Female BALB/c nude mice (body weight:~20 g) were purchased from Gempharmatech Co., Ltd. (Nanjing, China). All animal experiments complied with the Institutional Animal Care and Use Committee (IACUC). To establish the tumor model, HCT116 cells (1.0 × 10 6 cells) suspended in 25 µL McCoy s 5A and 25 µL Matrigel were inoculated in the right hind-leg region of the mice. The tumors were grown to 100-200 mm 3 before further application.

Ratiometric Fluorescence Imaging of Exogeneous H 2 S in Mice
To image the exogeneous H 2 S level in mice, saline (25 µL), NaHS (1 mM, 25 µL), or NaHS (1 mM, 25 µL) containing ZnCl 2 (1 mM) was subcutaneously injected into the right hind-leg region of the mice. Subsequently, HS-CG (20/20 µM IR-HS/ICG, 25 µL) in saline was subcutaneously injected into the mice. After 15 min, the fluorescence images of the mice were acquired on an IVIS Lumina XR III imaging system using a 660 nm excitation filter with a 730-790 nm emission filter for imaging IR-HS within HS-CG and a 780 nm excitation filter with n 825-865 nm emission filter for imaging ICG within HS-CG.

Ratiometric Imaging of Endogenous H 2 S Tumor of Mice
To image H 2 S in the HCT116 tumors, mice bearing subcutaneous HCT116 tumors were intravenously (i.v.) injected with HS-CG (40/40 µM IR-HS/ICG, 200 µL). After injection, the mice were i.t. injected with saline (25 µL) or L -Cys (1 mM, 25 µL) or L -Cys (1 mM, 25 µL) containing PAG (5 mg/kg) +AOAA (5 mg/kg). The fluorescence images were acquired at 12 h on an IVIS Lumina XR III imaging system using a 660 nm excitation filter with a 730-790 nm emission filter for imaging IR-HS within HS-CG and a 780 nm excitation filter with an 825-865 nm emission filter for imaging ICG within HS-CG.

Conclusions
In conclusion, we developed a ratiometric NIR fluorescence liposome nanoprobe (HS-CG) for H 2 S detection in vivo. We showed that HS-CG displayed fast kinetics toward H 2 S because DPPC that was positively charged could capture the anion HS − to increase the local concentration of HS − around HS-CG and improve the reaction rate between HS-CG and H 2 S. Doping positively charged DPPC into the nanoparticles could provide an effective strategy to improve the response toward H 2 S and enhance the detection sensitivity of H 2 S. HS-CG offers a turn-on NIR fluorescence ratio F 728 /F 816 that could be successfully applied to monitor H 2 S levels in living HCT116 cells and tumor H 2 S levels in mice, exhibiting a highly fast response that is specific toward H 2 S. Thus, the H 2 S-activatable ratiometric NIR fluorescence liposome nanoprobe has the potential to serve as an efficient imaging nanoprobe for the detection of H 2 S-related physiological and pathological processes in vivo. Institutional Review Board Statement: The study was approved by the local Institutional Animal Care and Use Committee (IACUC).

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.